ABSTRACT: We propose combination of the rabbit cornea endothelial and epithelial cells with bioengineered scaffolds in cell culture well inserts. This technology could significantly reduce the use of rabbits for the Draize rabbit eye irritation assay. The barrier to commercialization is the lack of an effective method for storing, banking, of living bioengineered cornea-equivalents. We will combine cell expansion and scaffold technology developed by our collaborator, Professor Soker at the Wake Forest Institute for Regenerative Medicine, with patented ice-free cryopreservation strategies developed by scientists at our company, Tissue Testing Technologies LLC. Cryopreservation using ice-free vitrification has shown promise with several human cell- derived construct models, including EpiOcular. This work will be performed in three specific aims: In the first aim a bioengineered gel scaffold originally formulated for human cells will be assessed for compatibility with rabbit endothelial and epithelial cells and optimized in culture well inserts. The scaffold and porous, clear insert membrane represents the stroma of the cornea. In the second aim we will compare three experimental groups, 1) corneal epithelial constructs without endothelial cells, 2) constructs with just an endothelium, and 3) cornea constructs with an endothelium under and an epithelium on top of the scaffold. They will be compared using established positive and negative control treatments to determine whether the less complicated constructs are more or less predictive of irritation than a construct with both endothelial and epithelial layers. In parallel with aim two the third aim will assess our lead ice-free cryopreservation strategy for short-term, 1-30 days, storage. During these studies a panel of biological and physical assays will be performed to determine the feasibility of employing our constructs for irritation evaluation of new chemical formulations. The Phase II plan will consist of further optimization, testing against a larger panel of irritants, long-term shelf-life evaluation and head-to-head comparison with the in vivo Draize rabbit eye test that will be required for translation, market acceptance, of cryopreserved bioengineered rabbit cornea-equivalents to the test market. !